**2. X-CHIP design and application**

The principal idea behind the X-CHIP was to create a platform that presents an alternative to the conventional crystallographic pipeline by consolidating the processes of crystallization condition screening, crystal inspection and data collection onto one device, streamlining the entire process and eliminating crystal handling and arduous cryogenic techniques (Fig. 1).

**Figure 1.** The X-CHIP was designed and developed as a miniaturized and integrated alternative to conventional methods of crystallization and data collection.

The chip is made from a material chosen for its visual light transparency and relatively low absorption of X-ray radiation. An X-CHIP with a thickness of 0.375 mm absorbed approximately thirty percent of the X-ray intensity of the primary synchrotron beam that was attenuated by 1,800 - 2,000 times to avoid excessive radiation damage to crystals during data acquisition. Designed to be compatible with most standard goniometers, the device inserts into a chip-base (possessing a machined slit and locking screw) for support and simple mounting (Fig. 1). A plastic receptacle holds multiple chips mounted on bases, providing rigidity for set up, storage and visual inspection of the crystallization drops and can be covered with a special lid to prevent dust contamination. The chip, along with supporting tools, is shown in Fig. 2.

88 Recent Advances in Crystallography

**2. X-CHIP design and application** 

techniques (Fig. 1).

The X-CHIP (Chirgadze, 2009) addresses the same challenges of high-throughput crystallography with an alternative approach, and has a number of unique additional advantages. In contrast to microfluidic chips, the crystallization process takes place on the chip surface, in droplet arrays of aqueous protein and crystallization reagents mixtures under a layer of oil. These microbatch arrays are made possible by altering the chip surface with a unique coating, creating defined areas of varying hydrophobicity. This paper presents the design of the device and accompanying tools for setting up crystallization trials and mounting the chip for data collection, as well as the important benefits, limitations and implications that are inherent to this platform. It also describes proof-of-concept experiments in which this technology was utilized for crystal growth, visual inspection, Xray diffraction data collection and structure determination of two native and one selenomethionine-labeled protein targets. The presented results show that large, welldiffracting crystals can be grown and high-quality data sets sufficient for structure

determination can be collected on a home as well as a synchrotron X-ray source.

The principal idea behind the X-CHIP was to create a platform that presents an alternative to the conventional crystallographic pipeline by consolidating the processes of crystallization condition screening, crystal inspection and data collection onto one device, streamlining the entire process and eliminating crystal handling and arduous cryogenic

**Figure 1.** The X-CHIP was designed and developed as a miniaturized and integrated alternative to

The chip is made from a material chosen for its visual light transparency and relatively low absorption of X-ray radiation. An X-CHIP with a thickness of 0.375 mm absorbed approximately thirty percent of the X-ray intensity of the primary synchrotron beam that was attenuated by 1,800 - 2,000 times to avoid excessive radiation damage to crystals during data acquisition. Designed to be compatible with most standard goniometers, the device

conventional methods of crystallization and data collection.

**Figure 2.** Schematics and images of the X-CHIP (*a*) Top-view schematic of the 24-drop format chip, hydrophilic and hydrophobic areas are shown in light and dark grey, respectively (other formats include 6-drops; not shown) (*b*) Cross-section of the chip (*c*) X-CHIP on a base and a 4-chip receptacle device (*d*) X-CHIP with 24 crystallization drops, mounted on a goniometer.

The described system applies principles of the microbatch crystallization method, the high effectiveness and unique benefits of which have been described elsewhere (D'Arcy *et al.*, 1996, Chayen, 1998, D'Arcy *et al.*, 2003). On the surface of the chip, circular hydrophilic areas are inscribed in hydrophobic annuli in ordered arrays (Fig. 2*a*, 2*b*). Nanoliter volumes of aqueous protein and precipitant solutions are mixed onto the hydrophilic circle by sequential addition and then covered by an mineral oil layer, which is dispensed on top of the drop and is stabilized on the surrounding hydrophobic ring. The interactions between the aqueous phase, oil layer and coated surface create highly defined droplets of predictable volume and thickness and prevent drops not only from drying but from fusing with each other with each other during crystallization set up and data acquisition. The design of the chip currently uses 1 x 6 and 4 x 6 formats and its size permits visual inspection of the entire chip in one image (Fig. 3*a*).

**Figure 3.** Experimental results: (*a*) section of a 4x6 X-CHIP with a two-dimensional optimization of two crystallization conditions for native PA0269, taken two weeks after initial set up (*b*) crystals of EphA3 grown overnight, crystal size approximately 250μm in length (*c*) On-the-chip diffraction image for an EphA3 crystal, collected on a Rigaku FR-E rotating anode with R-AXIS HTC detector (*d*) Part of an experimental electron density map generated using the SAD PA0269SM data set collected directly from the crystal grown on the X-CHIP, superimposed with the protein Cα-trace, shown as a black solid line.
